The present invention relates to a method for determining cholesterol, either total cholesterol or bound cholesterol, in human serum. More particularly, the invention relates to a method and stabilized enzymatic solutions for use in the method in determining the total cholesterol in serum.
Cholesterol is present in biological matter, such as serum and the like, partially in free form and partially in bound form as a cholesterol ester. For the determination of total cholesterol, it is necessary to release the cholesterol that is bound in cholesterol ester form. The releasing of the bound cholesterol has been conducted through saponification of the cholesterol ester under alkaline conditions using alcoholic potash lye, for example. Following the saponification, the released cholesterol can then be determined either chemically or enzymatically by one of the known methods. A chemical determination may be performed with or without saponification, for example, by the Liebermann-Burchard method. An enzymatic determination may be performed by means using cholesterol oxidase, cholesterol esterase, or cholesterol dehydrase.
The alkaline saponification of bound cholesterol is a troublesome and time consuming step in the overall assay of total cholesterol. Furthermore, the relatively aggressive reagents used may lead to a decomposition of the cholesterol. In order to prevent such decomposition and inhibit the determining of false and/or imprecise results of the analysis, a hydrolysis must generally be performed under relatively mild conditions. This, in turn, undesirably increases the length of time required for the cholesterol determination. The alkaline liberation of the bound cholesterol is especially disadvantageous when the determination of cholesterol is to be performed by the preferred enzymatic methods. Since the enzymes are inactivated in the strongly alkaline medium, the hydrolyzate must be neutralized by the addition of acid to a pH of about 5 to 8 before the enzymatic determination can be initiated. This extra step results in the addition of more time in the overall determination of the total cholesterol.
It is also known that bound cholesterol can be freed by the action of enzymes, cholesterol esterase, which break the ester bond in cholesterol esters. Such cholesterol esterases were isolated initially from animal sources, such as from pork pancreas and rat pancreatic juice. It is also known that in addition to cholesterol esterases being found in the pancreas, that cholesterol esterases can be found in the liver.
Allain, et al describe an enzymatic method for the determination of total serum cholesterol using a cholesterol esterase isolated from pork pancreas and rat pancreatic juice, "Clinical Chemistry," 20 (1974), 470-475. In the method of Allain, et al the cholesterol esterase (cholesterol ester hydrolyase), freed the esterified cholesterol. The resulting freed cholesterol was treated with cholesterol oxidase to form cholestenone and hydrogen peroxide. The resulting hydrogen peroxide was measured quantitatively using spectrophotometric methods. The hydrogen peroxide reacted with 4-aminoantipyrine and phenol in the presence of a peroxidase to form a quinoneimine dye. Allain, et al utilized one aqueous buffered solution to conduct the cholesterol determinations. Cholesterol esterases are known labile compounds that are generally unstable in aqueous solutions. Allain, et al state that the enzyme solution used in their method is unstable having a stability of eight hours at room temperature (25.degree. C.) and 24 hours at refrigerated temperatures of 4.degree. C.
U.S. Pat. No. 3,925,164 to Beauchamp, et al also describes a method for the enzymatic determination of total cholesterol in serum. The method therein treats the serum sample with a cholesterol esterase to release the bound cholesterol. The total cholesterol is then determined by known techniques. The method utilizes a cholesterol esterase produced from a microorganism rather than using a cholesterol esterase produced from an animal source. The patent states that the microorganism produced cholesterol esterase is preferred over cholesterol esterase produced from animal sources in the complete saponification of cholesterol esters in the framework of a quantitative analysis process because the cleavage rates for animal source cholesterol esterases were not quantitative. Furthermore, bound cholesterol is present in biological matter in the form of widely different acids. For an enzymatic process to be useful in the framework of a process of quantitative analysis, it is required that all of the esters that may occur be cleaved quantitatively with approximately the same speed and with the same reliability. Many of the known animal-source cholesterol esterases are somewhat specific toward specific cholesterol esters. The activity of such cholesterol esterases is known to vary with regard to various cholesterol esters.
Although the cholesterol esterases produced from microorganisms offer advantages over those produced from animal sources, the microorganism-produced cholesterol esterases also are labile compounds that tend to undergo chemical change in solution and especially in aqueous solutions, which decreases their enzymatic activity. Stability of enzymatic solutions used in diagnostic assays is important in providing methods of analysis which exhibit precision and uniformity among separate determinations when conducted over a period of elapsed time. Instability of enzymatic solutions, in addition to not providing reproducibility of assays, can also add to the ever increasing cost of medical services because the unstable enzymatic solutions need to be discarded and fresh solutions formulated.
It has recently been estimated that about 25 percent of all in vitro diagnostic tests conducted annually in the United States are unreliable. Unreliable tests can result in unnecessary medical treatment, the withholding of necessary treatment and lost income. Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years and indications are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement derives from the fact that the exact nature of enzymes, as well as mechanisms of their reactions, remains unknown for the most part.
At present, the greatest limitation in the diagnostic reagent manufacture, by far, lies in the unstable characteristics of the enzymatic solutions. Current cholesterol diagnostic methodologies require the use of labile ingredients whether utilizing enzymes from microorganisms or animal source. Due to the labile nature of the enzymes, rigorous quality control is required over the production of such enzymatic solutions and in reconstituting dry media preparations and formulation of such enzymatic solutions. Such quality control is costly. Moreover, if such control in any step in the process is not maintained within a high degree of control standards, the quality of the final product can be reduced materially leading to decreased precision in assay results.
The present commercial state-of-the-art used for stabilizing the reactive ability of enzymes or coenzymes is by locking them into a solid matrix, either by freeze drying, dry blending such as used for tableting dry powders primarily in the pharmaceutical diagnostic and related industries, and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending. Usefulness of the product is further limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to achieve, especially in the laboratories where the products are to be utilized in diagnostic assay. This condition is exemplified by the fact that most commercial freeze-dried controlled sera (reference serum) lists the acceptable bottle-to-bottle variation of enzyme constituents at .+-.10 percent of the mean.
The present invention is uniquely designed so that the enzyme solution, although containing labile ingredients in a liquid reagent, are effectively "stabilized" thereby controlling the activity of the labile ingredients in the liquid solution. The means of stability insures long-term stability in a liquid media. Moreover, close tolerance control can be achieved in the manufacturing of a high quality product which eliminates the inconvenience of the rigid package size, the high cost of packaging and freeze drying, and reagent waste.